(2&#39;-5&#39;)-Oligo (3&#39;-deoxyadenylate) and derivatives thereof

ABSTRACT

3&#39;-Deoxyadenosine 5&#39;-triphosphate is oligomerized to form (2&#39;-5&#39;)-oligo (3&#39;-deoxyadenylate) by incubation with adenosine triphosphate: (2&#39;-5&#39;)-oligo adenosine adenyl transferase, in the presence of an inert support carrying a double stranded polynucleotide. The (2&#39;-5&#39;)-oligo (3&#39;-deoxyadenylate) is digested with a suitable phosphatase to remove the terminal phosphate groups. The thus produced corresponding 3&#39;-deoxyadenosine compound is an anti-viral material effective against Herpes Simplex infection and effective in inhibiting the transformation of cells infected with Epstein Barr virus.

BACKGROUND OF THE INVENTION

1. Field of the Invention

There are provided oligomers of 3'-deoxyadenosine-5'-triphosphate andthe terminally dephosphorylated analogs thereof which are usefulanti-viral agents and methods for producing said materials.

2. Discussion of the Relevant Art

The full nomenclature of the subject matter of the present inventioninvolves extremely long terms. It is customary for those skilled in theart to abbreviate these terms in a manner well known to them. Thesegeneral and customary abbreviations are set forth herein below and willbe utilized in the text of this specification.

Abbreviations: EBV, Epstein Barr virus; (2'-5')-(A)_(n) synthetase, ATP:(2'-5')oligo(A) adenyltransferase (EC 2.7.7.-); (2'-5')pppA(pA)_(n) or(2'-5')oligo(A), oligomer of adenylic acid with (2'-5')-phosphodiesterlinkages and a triphosphate at the 5'-end; (2'-5')ppp3'dA(p3'dA)_(n),oligomer of 3'-deoxyadenylic acid with (2'-5')-phosphodiester linkagesand a triphosphate at the 5'-end; (2'-5')A(pA)_(n) or core oligomer,oligomer of adenylic acid with (2'-5')-phosphodiester linkages;(2'-5')A(pA)₂ or core trimer, adenylyl-(2'-5')adenylyl(2'-5')adenosine;(2'-5')A(pA)₃ or core tetramer, adenylyl(2'-5')adenylyl(2'-5')-adenylyl(2'-5')adenosine; (2'-5')3'dA(pB' dA)₂ or core trimer analog,3'-deoxyadenylyl (2'-5')3'-deoxyadenylyl(2'-5')3'-deoxyadenosine;poly(rI), poly(rC)-agarose, poly(inosinate).poly(cytidylate) doublestranded polynucleotide covalently bound to agarose though poly(rI);HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; A, adenosine;3'dA, 3'-deoxyadenosine (3'-deoxyadenylate); pA, 5'-AMP; p3'dA,3'-deoxy-5'-AMP or 3'-deoxyadenylate; 3'-dATP, 3'-deoxy-ATP; BAP,bacterial alkaline phosphatase; SVPD, snake venom phosphodiesterase I;ATP; a denosine triphosphate; AMP, adenosine monophosphate.

Under ordinary circumstances a cell will contain ATP and ATP synthetaseand an endonuclease which will not interact. In a manner not entirelyunderstood where viral infection of the cell is about to occurinterferon reacts upon the cell causing the synthetase to act upon theATP to form (2'-5')-oligo(A) having the general designation (2'-5')p_(m)A(pA)_(n) where m is 1,2 or 3 and n may be 1-14, preferably 1-5. Uponintrusion of the virus which contains double stranded ribonucleic acid,the (2'-5') oligo (A) activates the endonuclease which then destroys themessenger ribonucleic acid of the virus, preventing replication of thevirus. In this anti-viral defense mechanism of the cell, generallyspeaking, the (2'-5') oligo (A) produced by the cell, is destroyed. Thehalf life of this oligomer is extremely short and cannot therefore beutilized as an externally administered anti viral material without beingimmediately destroyed by the cell defense mechanisms. It would bedesirable therefore to provide an endonuclease activator ofsubstantially greater half life than the (2'-5') oligo (A) which can beadministered as a anti-viral material without being immediatelydestroyed by the cellular defense mechanisms.

The existance of the (2'-5') phosphodiester bond in nature is an item ofcomparatively recent knowledge (Cory et al Biochim Biophys Acta 103, 646(1965)).

In classical work by Lord Todd it was shown that in the more commonlyrecognized (3'-5') phosphodiester bond compound a cyclic linkage betweenthe 3'-hydroxyl group and the bridging phosphate group between the tworibose moieties was essential for the hydrolytic cleavage of thephosphodiester linkage. It is further known that all reactions involvingadenosine triphosphate depend upon the presence of an hydroxyl moietyeither in the 2'-or in the 3'-position of the ribose nucleus of theadenosine moiety. In considering the reasons for the rapid destructionof (2'-5') oligo (A) in the cell it will be seen that the 2'-positioncan not participate since it does not bear a hydroxyl group.

The inventors herein therefore postulated that since the 2'-position wasnot available for reaction, the degradation of the oligomer mightproceed via a cyclic mechanism involving the 3'-hydroxyl in a mannersimilar to the involvement of the 2'-hydroxyl as shown by Todd. Theinventors herein therefore further postulated that the absence of ahydroxyl moiety at the 3'-position might interupt the hydrolyticdegradation of the (2'-5') phosphodiester linkage and thus lead to anincreased half life for the resulting product. They further postulatedthat if the presence of the aforesaid 3'-hydroxyl group was notessential for the activation of the endonuclease there might be provideda material which would carry out the endonuclease activating function of(2'-5') oligo (A) while having a substantial greater half-life.

Primary exposure to Epstein Barr virus (EBV) in childhood usuallyresults in a silent infection. If infection with EBV is postponed untiladolescence, a more serious illness, infectious mononucleosis, developsin approximately 40 percent of the individuals. Although usually thoughtof as resulting in a self-limiting lymphoproliferative disease, it isbecoming increasingly clear that primary infection with EBV can resultin life-threatening illness in both adolescents and children. This factadds support that the morbidity and mortality associated with EBVinfection is more serious than originally thought. EBV infection hasalso been associated with several malignancies, Burkitt's lymphoma andnasopharyngeal carcinoma and Hodgkin's disease.

In vitro systems have been used to evaluate the ability of compoundswith anviviral properties to inhibit EBV replication, including1-β-D-arabinofuranosylthymine and 9-β-D-arabinofuranosyladenine. One ofthe difficulties encountered with nucleoside analogs as antiviral agentsis specificity for the virus-infected cell and concomitant cytotoxicity.Another difficulty with nucleoside analogs (such as9-(2-hydroxyethoxymethyl)guanine and 5'-amino-5'-deoxyiodouridine) asantiviral agents is that they can act only after the mammalian cell isinfected by a virus and the viral deoxythymidine kinase is expressed.Because of these difficulties with the use of nucleosides, interest hasgrown in the use of autochthonous interferon as an antiviral andantineoplastic agent. However, the development and use of interferon asan antiviral agent has been limited due to scarcity and problemsassociated with delivery to target cells. Another disadvantage ofinterferon as an antiviral/antitumor agent is the recent report of theformation of antibodies to interferon. Few in depth in vitro studieshave been reported on the effects of purified interferon on EBVreplication in human lymphocytes, although fibroblast transformation byoncogenic DNA viruses has been successfully inhibited by exogenouslyadded interferon. It has recently reported that the transformation ofadult, but not newborn, lymphocytes by EBV and phytohemagglutinin isinhibited by interferon.

Interferon-treated cells develop the antiviral state by the induction of(2'-5')(A)_(n) synthetase and a protein kinase. The (2'-5')A(pA)_(n)formed by the synthetase in the presence of double stranded RNAactivates a latent endoribonuclease which degrades messenger RNA. Thusit appeared to the inventors herein that Epstein Barr virus wouldprovide an interesting test model for the aforementioned hypotehesis.

SUMMARY OF THE INVENTION

(2'-5')-Oligo (3'-deoxyadenylate), an analog of (2'-5')-oligo (A)possessing the general structure p_(m) 3'dA[2'p5'(3'dA)]_(n) where m is1,2, or 3 and n is 1,2,3, or 4 is synthesized by incubating 3'-dATP witha suitable ATP: (2'-5')oligo(A) adenyltransferase in the presence ofcertain double stranded polynucleotides covalently bound to a supportphase. The products of the incubation are purified by chromatographicmeans. The major products are the trimer triphosphates (m=3, n=2) thetetramer diphosphate (m=2, n=3) and the tetramer triphosphate (m=3,n=3), and the dimer triphosphate (m=3, n=1) the trimer inhibit proteinsynthesis in lysed rabbit reticulocytes.

The (2',5')-oligo (3'-deoxyadenylate) was incubated with a phosphataseeffective against terminal phosphate groups but inert with respect tophospha diester groups suitably bacterial alkaline or acid phosphataseto provide the corresponding core oligomer analog that is to say, the3'-deoxyadenosine in place of the 3'-deoxyadenosine polyphosphate.

The core analog oligomer has been found to inhibit the transformation ofEpstein Barr virus in human lymphocytes in the absence of interferon andhas similarly been found to inhibit replication of Herpes Simplex virustype 1 in human fibroblast monolayers, also in the absence ofinterferon.

It is the surprising finding herein that while the core analog oligomer(2'-5')3'dA(2'p5'3'dA)₃ has the foregoing transformation inhibitoryactivity as does the natural core oligomer (2'-5')dA(2'p5'dA)_(n) incontrast to the latter, the former has been found to be non-cytotoxic atthe levels tested as well as having a substantially greater half life.

The compounds of the present invention that is to say, compounds havingthe structure: ##STR1## may be taken up in pharmaceutically acceptablecarriers for example, solutions, suspensions, tablets, capsules,ointments, eloxias, and injectable compositions and the like andadministered to subjects suffering from viral infection the dosageadministered depending upon the severity of the infection and the sizeand weight of the infected subject. The compositions may particularly beemployed against Epstein Barr virus and herpes simplex infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of DEAE chromatographic elution ofthe enzymic oligomerization product with markers.

FIG. 2 is a stability representation of (2'-5')p₃ A(pA)_(n) and(2'-5')p₃ 3'dA(p3'dA)_(n) in HeLa cell extracts.

FIG. 3 is a graphical representation of inhibition of Leucinetranslation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the process of the present invention 3'-dATP is incubated with asuitable double stranded polynucleotide covalently bounded to a suitablesubstrate in the presence of an appropriate ATP: (2'-5')oligo(A)adenyltransferase.

The transferase may be isolated from a number of sources suitably fromlysed rabbit reticulosites, L cells and HeLa cells. For the transferaseto be effective it is important that it be bound to a double strandedpolynucleotide. It has been found that poly(rI).poly(rC) in columnwherein the material is bound to the substrate, suitably agarose,through the poly(rI). is especially suitable.

Where the transferase is derived from HeLa cells there is utilized theprocedure of Baglioni et al (Biochemistry 18, 1765-1770 (1979)). Where Lcell extract is utilized this is prepared in accordance with the methodof Lenz et al (Biochemistry 17, 87 (1978)), similarly, when LRR (lysedrabbit reticulocyte) is utilized.

The column wash buffer comprises aqueous potassium chloride, aqueousmagnesium acetate, aqueous dithiothreitol, glycerol and HEPES to pH 7.5.The 3'-dATP in the same buffer is charged to the column and the columnincubated. Suitably, incubation is carried out at between 25° and 35°C., suitably at about 30° C., for from between 5 to about 25 hours. Ithas been found that incubation periods exceeding 15 hours are desirablefor a reasonable level of conversion. Longer incubation periods have sofar not been found to be harmful or leading to degradation of product.If desired, radioactive marker compounds may be utilized to assist inindentification of the desired fraction. For this purpose [8-³ H] or[α-³² P] may be utilized as the radioactive markers. A radioactivity ofthe order of between 500 and 5000 mCi/m mole may be employed for thetritium marker and between 500 and 5000 Ci/m mole for the phosphorusmarker. The column is then eluted with the above-mentioned column washbuffer. The product is then applied to a DEAE cellulose column andwashed therefrom with aqueous potassium chloride suitably between 0.1and 1 molar. If desired the product may be further purified bychromatography on Sephadex suitably Sephadex G-25.

There is thus obtained the desired p_(m) 3'dA[2'p5'(3'dA)]_(n) where mis 1,2 or 3 and n is 1,2,3 or 4.

In order to produce the "core analog" the aforementioned oligomer isdephosphorylated at the 5'-position. There may be utilized anyphosphatase or combination of phosphatases effective against terminalphosphate groups but inert with respect to phosphodiester groups. Thus,the phosphate groups at the 5'-position would be removed while thephosphate groups bridging between the 2' and 5'-positions of theadjacent ribose nuclei would be unaffected. There may be utilizedbacterial alkaline or acid phosphatase of which bacterial alkalinephosphatase is especially preferred. The procedure, however, is notrestricted thereto. While BAP will remove all of the phosphate groups atthe 5' position this removal may, if desired, be carried out in twostages. Thus, utilizing apyrase, ATPase, pyrophosphatase the outermosttwo phosphate groups of a triphosphate moiety or the outermost phosphategroup of a diphosphate moiety may be removed.

The final phosphate group may then be removed with any phosphatase orany 5'-nucleotidase. In the preferred procedure the (2'-5')oligo(3'-deoxyadenylate) is digested with BAP in a suitable buffer forexample, Tris hydrochloride at pH of about 8 for from about 45 to about120 minutes at from about 30° to about 40° suitably about 37° C. Thereaction is terminated by the addition of aqueous urea and the reactionmixture is purified on Sephadex and lyophilized.

Several experiments were done to characterize the structure of theputative (2'-5')ppp3'dA(p3'dA)₂ and to establish its biologicalproperties. Treatment of the putative [³² P](2'-5')ppp3'dA(p3'dA)₂ withBAP followed by XAD-4 chromatography resulted in the isolation of 34% ofthe ³² P as ³² P_(i) (theoretical--33.3%). Additional proof for thestructure of (2'-5')-ppp3'dA(p3'dA)₂ was obtained by treatment of theadenylate analog with BAP, SVPD I, and T2 RNase followed by tlc andmeasurement of radioactive compounds. The nucleotide synthesized from[α-³² P]3'dATP and purified by DEAE cellulose chromatography (FIG. 1,charge--minus 6) showed only one radioactive spot (FIG. 1,).

BAP hydrolysis of the putative [α-³² P](2'-5')-ppp3'dA(p3'dA)₂ produced³² P_(i) and core [³² P](2'-5')-3'dA(p3'dA)₂. There was no ³² P in theregion of (2'-5')A(pA)₃. Therefore, the putative [³² P]ppp3'dA-(p3'dA)₂was not contaminated with (2'-5'(ppA(pA)₃ (charge--minus 6). Hydrolysisof core [³² P]3'dA(p3'dA)₂ with SVPD I formed [³² P]3'dAMP; there was no³² P in the AMP region. Therefore, the [α-³² P]3'dATP could not becontaminated with [α-³² P]ATP.

Further proof of the 2',5' linkage was obtained by treatment of theputative (2'-5')ppp3'dA(p3'dA)₂ by T2 RNase; there was no hydrolysis.Core [³² P] (2'-5')-3'dA(p3'dA)₂, [³² P]3'dAMP, and [³²P](2'-5')ppp3'dA(p3'dA)₂ following elution and rechromatography oncellulose tlc plates in solvent B showed only one radioactive spot each.

The structural characterization is supported by further experiments onthe core trimer analog produced by BAP digestion.

On the aforementioned DEAE chromatography, the putative(2'-5')3'dA(p3'dA)₂ ran with AMP (charge -2) indicating it too had acharge of -2, which would be correct. The SVPD digestion carried out on[G-³ H]-(2'-5')3'dA(p3'dA)₂ showed not only the expected 3'dAMP but alsothe expected 3'dA, though the latter was not isolated in thequantitatively expected amount.

All references herein cited with respect to synthetic or analyticalprocedures are incorporated herein by reference.

EXAMPLES EXAMPLE I: Cell Culture and Virus Preparation

Heparinized whole venous blood was obtained from the umbilical cords ofnewborn infants (human umbilical cord lymphocytes) or EBV-seronegativevolunteers (peripheral blood lymphocytes) in collaboration with TempleUniversity Hospital, Philadelphia, PA. Mononuclear leukocytes wereprepared by the Ficoll-Hypaque method of Boyum (Scand. J. Clin. Invest.21 (Sup. 97), 77 (1968)) and were cultured in RPMI-1640 supplementedwith 20% fetal bovine serum as described (Henderson et al, Virology 76,152 (1977)). The lymphoblastoid cell lines BJAB and RAJI were gifts ofDrs. Werner and Gertrude Henle, Children's Hospital, University ofPennsylvania, Philadelphia, PA, and were cultured as described above.The lymphoblastoid line C85-5C was obtained by transformation of humanumbilical cord lymphocytes following infection with EBV and maintainedas described aove. Cultured mouse L cells were maintained in minimumessential medium (Eagle) supplemented with Earle's salts, L-glutamineand 6% fetal bovine serum. Interferon-treated mouse L cells weresupplied by Dr. Esther H. Chang, National Cancer Institute, NIH. EBVstocks produced from the cottom top marmoset line B95-8, obtained fromDr. George Miller, Yale University, had a TD₅₀ /0.20 ml of equal to orgreater than 10⁴ as determined on human umbilical cord lymphocytes. EBVstocks were maintained at -70° C. until use. Human umbilical cordlymphocytes and peripheral blood lymphocytes were infected as described(Henderson et al, Supra).

EXAMPLE II: Enzymatic synthesis and isolation of(2'-5')ppp3'dA(p3'dA)_(n) in L cell extracts

Interferon treated L cell extracts were prepared as described by Lenz etal, Biochemistry 17, 80 (1978). Poly(rI). poly(rC)-agarose, packed in asmall column (0.5 ml), was washed with 50 ml of buffer A (90 mM KCl, 1.5mM Mg(OAc)₂, 1 mM dithiothreitol, 100 mM HEPES, pH 7.5, 10% glycerol).Interferon-treated L cell extracts (1 ml) were passed through the columnand washed with 80 ml of buffer A. A reaction mixture containing 50 mMMg(OAc)₂, 1 mM dithiothreitol, 90 mM KCl, 10 mM HEPES, pH 7.5, 10%glycerol, 5 mM [α-³² P]3'dATP (Amersham/Searle; 2μ Ci; 3000 Ci/mmol) wasadded to the column and incubated for 24 hr at 42° C. One ml of waterwas added to displace the nucleotides. The water was removed bylyophilization and unreacted 3'dATP was converted to 3'dADP byhexokinase (type VII, Sigma Chemical Co.) as described by Dougherty etal, J. Biol. Chem. 255 3813 (1980). The reaction products werechromatographed on PEI-cellulose tlc plates (Polygram CEL 300 PEI,Brinkman) in 0.75M potassium phosphate, pH 3.5. One major radioactivearea corresponding to 3'dADP was detected. A second radioactive areacorresponding to a slower migrating compound was detected andtentatively identified as trimer analog. The putative radioactive trimeranalog was eluted from the plate with 1M NH₄ HCO₃, lyophilized, andreconstituted in glass distilled water.

EXAMPLE III: Oligomer Synthesis

Poly(rI). poly(rC)-agarose columns (0.5×1.5 cm) bound with (2'-5')(A)_(n) synthetase were prepared as described by Baglioni et al(Biochemistry 18, 1765 (1979)), except that reticulocyte lysate (1.2 ml)was used in place of HeLa cell extract and the column wash bufferconsisted of 100 mM KCl, 2 mM Mg(OAc)₂, 2 mM dithiothreitol (DTT), 10%glycerol and 20 mM HEPES, pH 7.5 (buffer A). The final concentration ofMg(OAc)₂ was 8 mM in all incubations except for the [G-³ H]3'dATPexperiment where the concentration was 2.3 mM. Columns were incubated at30° C. for the time periods indicated with [8-³ H]ATP (10 Ci, 22 Cimmol⁻¹), [α-³² P]3'dATP (25 Ci, 640 or 3000 Ci mmol⁻¹), or [G-³3H]3'dATP (10μ Ci, 640 mCi mmol⁻¹). Ninety five percent of theradioactivity was eluted with 2 ml of buffer A. [α-³² P] 3'dATP waspurchased from Amersham Corporation (Arlington Heights, IL,) and newEngland Nuclear Corporation (Boston, MA,). Cordycepin,(3'-deoxyadenosine), was isolated and purified from Cordyceps militaris(mp 228°-229° C., literature 231° C.). Commercially available 3'dATP ofthe highest purity was purchased from Sigman Chemical Co. (St. Louis,MO,) or synthesized (Suhadolnik et al, J. Biol. Chem. 252 4125 (1977)).[G-³ H]3'dATP was synthesized in this laboratory, oxidized withperiodate to remove contaminating ATP or other ribonucleotides andrepurified by descending paper chromatography (Whatman 3MM in solvents Aand B; solvent A: isobutyric acid-concentrated ammonia-water, 66:1:33,v/v/v; solvent B: 1-propanol-concentrated ammonia-water, 60:30:10,v/v/v). In solvents A and B, one uv absorbing region or one radioactiveregion was detected for either 3'd-ATP, [G-³ H]3'dATP or [α-³² P]3'dATP.The ratio of tritium in the adenine:3-deoxyribose of the [G-³ H]cordycepin was 9:1. Product formation was determined by the amount ofradioactivity displaced from the DEAE cellulose column with 0.35M KCldivided by the total radioactivity recovered, except for the [G-³H]-3'dATP experiment. Percent conversion for the [G-³ H]3'dATPexperiment was determined from the elution profile shown in FIG. 1 asthe percentage of radioactivity eluting after 3'dATP becausechromatography of this material on DEAE cellulose and elution with 0.35MKCl was not performed prior to the DEAE cellulose chromatography NaClgradient elution method shown in FIG. 1. The amount of ³² P displacedwith 0.35M KCl when 25μ Ci of [α-³² P]3'dATP was added to DEAE cellulosecolumns was 0.15%, which was subtracted from the ³² P in the 0.35M KClfraction.

The total product obtained comprises product where m=3 and n=1 and 2predominantly.

In accordance with the above procedure oligomers wherein n=3 or 4 mayalso be obtained in lesser amounts.

Chromatography of the analog, [³ H](2'-5')p_(m) 3'dA-(p3'dA)_(n), fromincubations with [G-³ H]3'dATP on DEAE cellulose

Peak fractions of material washed from poly(rI).poly(rC)-agarose columnsfrom a 17 h incubation with [G-³ H]3'dATP were applied to a DEAEcellulose column (Whatman DE52, 0.5×17 cm). The nucleotides weredisplaced with a 50-150 mM linear gradient of NaCl (40 ml/40 ml), 50 mMtris-HCl, pH 8.0 in 7M urea; 1 ml fractions were collected; flow rate: 4ml h⁻¹. Cordycepin (3'dA), 3'dAMP, 3'dADP, 3'dATP, and authentic(2'-5')pppA(pA)₂ were included as markers and were monitored on arecording spectrophotometer (LKB 2138 Uvicord S). In the otherincubations with [α-³² P]3'dATP and [8-³ H]ATP, the radioactive materialeluting from the poly(rI).poly(rC)-agarose columns was chromatographedon DEAE cellulose columns (0.6×2.0 cm) and washed with 42 ml of bufferB: 0.09M KCl, 20 mM HEPES, pH 7.5. The oligonucleotides were displacedwith 5 ml of buffer B supplemented with 0.35M KCl. The radioactivenucleotides in the 0.35M KCl were further fractionated by the 7Murea--NaCl gradient--DEAE cellulose chromatography method describedabove.

The peak for (2'-5')p_(m) 3'dA(p3'dA)_(n) where m=3, n=1 precedes thatwhere m=3, n=2, the latter running at the same rate as the corresponding3'-hydroxylated oligomer.

In accordance with the above procedure, but where, in place of L cellextracts there are utilized HeLa cell extracts, the same product isobtained.

                  TABLE 1                                                         ______________________________________                                        Enzymatic Synthesis of (2'-5')p.sub.m 3'dA(p3'dA).sub.n                       and (2'-5')pppA(pA).sub.n                                                                                     Conversion to                                            Final      Incubation                                                                              (2'-5')                                                  concentration                                                                            time      oligonucleotide                               Substrate  (mM)       (h)       (%)                                           ______________________________________                                        [8-.sup.3 H]ATP                                                                          2           5        21.9                                          [8-.sup.3 H]ATP                                                                          2          17        19.0                                          [α-.sup.32 P]3'dATP                                                                2           5        0.3                                           [α-.sup.32 P]3'dATP                                                                2          17        3.0                                           [G-.sup.3 H]3'dATP                                                                       1.3        17        3.7                                           ______________________________________                                    

EXAMPLE IV: Enzymatic Dephosphorylation of (2'-5')p_(m) 3'dA(p3'dA)_(m)

(2'-5')p₃ 3'dA(p3'dA)₃ (1.2 μM) were digested with bacterial alkalinephosphatase (0.28 mg) in Tris hydrochloride (58 mM, 0.5 ml, pH 8.0) for90 minutes at 37° C. The digestion was terminated by the addition ofurea (7M, 3 ml) and the reaction mixture washed through a sephadex G10column with buffer A. The elution was followed by UV analysis at 260 nMand the peak material lyophylised to yield the desired(2'-5')-3'dA(p3'dA)₃. In accordance with the above procedure, but wherem is 1 or 2 and n is 1, 2, 3 or 4, there is obtained the corresponding5'-dephosphorylated oligomer having an unchanged n value respectively.

In accordance with the foregoing procedure, but where, in place ofbacterial alkaline phosphatase, there is utilized bacterial acidphosphatase at pH 8, there are obtained the same products.

In accordance with the foregoing procedure, but where, in place ofbacterial alkaline phosphatase, there is utilized apyrase, ATPase, orpyrophosphatase, there is obtained the corresponding(2'-5')p3'dA(p3'dA)_(n) further digestion with 5'-nucleotidase or anyphosphatase there is obtained the corresponding 5'-dephosphorylatedoligomer.

EXAMPLE V: Characterization of (2'-5')ppp3'dA(p3'dA)_(n)

[³² P](2'-5')-ppp3'dA(p3'dA)_(n) chromatographically pure, showedresistance to hydrolysis by T2 ribonuclease which is evidence for the(2'-5') linkage. Treatment of [³² P](2'-5')ppp3'dA(p3'dA)_(n) withbacterial alkaline phosphatase produced ³² P_(i) and core trimer analog((2'-5')3'dA(p3'dA)_(n)). No radioactivity was detected in the coretrimer region. Snake venom phosphodiesterase treatment of [³²P](2'-5')ppp3'dA(p3'dA)_(n) yielded only [³² P]3'dAMP. No radioactivitywas detected in the AMP region. At 100 nM the trimer analog inhibitedtranslation 80% in lysed rabbit reticuloytes, while the inhibition by100 nM authentic trimer was approximately 20%.

BIOLOGICAL ACTIVITY

The inhibition of translation by the analog, (2'-5')ppp3'dA(p3'dA)₂, wascompared with the inhibition by tetramer adenylate, (2'-5')pppA(pA)₃,and the trimer adenylate, (2'-5')pppA(pA)₂. The inhibition oftranslation by 40 nM (2'-5')pppA(pA)₃ was 52% and decreased to controlvalues at 0.004 nM; the inhibition of translation by 22 nM(2'-5')ppp3'dA(p3'dA)₂ was 68% and decreased to control values at 0.04nM. The trimer analog, (2'-5')ppp3'dA(p3'dA)₂, is about four times morepotent an inhibitor of translation than is (2'-5')pppA(pA)₂.

The stability of (2'-5')ppp3'dA(p3'dA)_(n) in HeLa cell extracts wascompared to that of (2'-5')pppA(pA)_(n). There was no hydrolysis of theanalog, (2'-5')ppp3'dA(p3'dA)_(n) (12 μM), by HeLa cell extractsfollowing a 45 min incubation; however, the t_(1/2) of(2'-5')pppA(pA)_(n) (5 μM) is about 2 min (FIG. 2). With 500 μM(2'-5')pppA(pA)_(n), there was a 50% hydrolysis in 45 min as determinedby DEAE cellulose chromatography; with 500 μM authentic core analog,(2'-5')3'dA(p3'dA)₂, there was no hydrolysis as determined by cellulosetlc (solvents A and B). Neither cordycepin nor 3'dAMP was detected. Thelack of hydrolysis of the analog may be similar to the lack ofhydrolysis of (3'-5')2'-deoxyoligonucleotides by the2',5'-phosphodiesterase.

The antiviral properties of 5'-dephosphorylated "core" (2'-5')A(pA)₂,(2'-5')A(pA)₃ and (2'-5')3'dA-(p3'dA)₂ were compared to the antiviralproperties of IFN-α and IFN-β. The (2'-5')oligonucleotides andinterferons inhibited Epstein-Barr Virus (EBV)-induced stimulation ofDNA synthesis in EBV infected human lymphocytes. Whereas (2'-5')A(pA)₂and (2'-5')A(pA)₃ were potent inhibitors of DNA synthesis of infectedand uninfected lymphocytes, (2'-5')3'dA(p3'dA)₂ inhibited onlyviral-induced DNA synthesis. The "core" 3'-deoxy (2'-5') oligonucleotideanalog inhibited EBV-induced morphological transformation of lymphocytesmuch the same as "core" (2'-5')A(pA)₂ and (2'-5')A(pA)₃. (2'-5')-A(pA)₂(300 μM) and (2'-5')A(pA)₃ (150 μM) were toxic to the lymphoblastoidlines Raji and BJAB whereas no toxicity was observed for(2'-5')3'dA(p3'dA)₂ (determined by inhibition of colony formation).

Effects pon IFN-β and (2'-5')Oligonucleotides of Herpes Simplex VirusType 1 (HSV-1) Replication

(2'-5')- and (3'-5')A(pA)₃ have been reported to be taken up by intactlymphocytes. Therefore, we employed an infected centers assay utilizinglymphocytes to determine the ability of (2'-5') oligonucleotides toinhibit HSV-1. After incubation for 72 hr with or without(2'-5')oligonucleotides, leukocytes exposed to HSV-1 were plated ontohuman foreskin fibroblast monolayers (Table 2). Leukocytes not treatedwith IFN-β or (2'-5')oligonucleotides developed as infected centers,whereas the fibroblast monolayers onto which IFN-β-treated leukocyteswere plated developed significantly less cytopathic effects and thenonly when higher (>67×10⁵ infected cells) were plated onto thefibroblast monolayers. Monolayers exposed to uninfected fibroblasts didnot develop cytopathic effects (cpe). (2'-5')A(pA)₂ and (2'-5')-A(pA)₃inhibited the development of cpe, with significant cpe observed onlywith fibroblasts treated with 2×10⁵ infected cells. (2'-5')3' dA(p3'dA)₂was less effective in inhibiting the development of cpe from infectedleukocytes compared to IFN-β, (2'-5')A(pA)₂, and (2'-5')A(pA)₃.

                  TABLE 2                                                         ______________________________________                                        Effect of IFN-β and (2'-5')oligonucleotides on development of            infected centers from HSV-1 infected leukocytes as                            determined by cytopathic effects (cpe) using human foreskin                   fibroblasts as indicators                                                                   cpe at given number of                                                        HSV-exposed leukocytes per cell                                 Treatment       2 × 10.sup.5                                                                     6.7 × 10.sup.4                                                                    2.2 × 10.sup.4                       ______________________________________                                        None (control)  +++      ++        +                                          IFN-β (25 U/ml)                                                                          ++       +         0                                          (100 U/ml)      +++      0         0                                          (250 U/ml)      +        0         0                                          (500 U/ml)      0        0         0                                          (2'-5')A(pA).sub.2 (10 μM)                                                                 +++      ++        0                                          (50 μM)      ++       ++        0                                          (75 μM)      ++       0         0                                          (150 μM)     +        0         0                                          (2'-5')A(pA).sub.3 (10 μM)                                                                 ++       +         0                                          (50 μM)      +        0         0                                          (75 μM)      ++       0         0                                          (150 μM)     +        0         0                                          (2'-5')3'dA(p3'dA).sub.2 (10 μM)                                                           +++      ++        +                                          (50 μM)      ++       ++        +                                          (75 μM)      ++       +         0                                          (150 μM)     --       --        0                                          (3'-5') A(pA).sub.2 (300 μM)                                                               +++      0         0                                          ______________________________________                                         +++ = complete destruction of the monolayer                                   ++ = multiple foci of cpe                                                     + = one focus of cpe                                                          0 = no monolayer disturbances                                            

Effect of Interferon and (2'-5')Oligonucleotides on Lymphoblastoid CellSurvival

IFN-α, core trimer, core tetramer, and core pentamer have been reportedto inhibit DNA synthesis in concanavalin A stimulated mouse spleenlymphocytes. Therefore, the effect of these agents on colony formationin closely related lymphoblastoid cell lines was determined (Table 3).IFN-β and IFN-B had no appreciable effect on lymphoblast colonyformation. (2'-5')A(pA)₂ at high concentrations (150-300 μM) and(2'-5')A(pA)₃ (50-100 μM) were found to be toxic to BJAB and Rajilymphoblasts. However, the (2'-5')2'dA(p3'dA)₃ was not cytotoxic toeither BJAB or Raji lymphoblasts at concentrations up to 300 μM. Thenatural (3'-5')A(pA)₂ also found non-cytotoxic does not inhibittransformation either and can be classed as inactive. Therefore, basedon the concentrations of (2'-5')oligonucleotides required to producecytotoxicity in lymphoblasts, the inhibition of EBV-inducedtransformation by interferon, core trimer, core trimer analog, and to alesser extent, core tetramer, was not the result of lymphocyte killingby these compounds.

                  TABLE 3                                                         ______________________________________                                        Cytotoxic effect of IFN-α, IFN-β, and                              oligonucleotides on established lympho-                                       blastoid cell lines*                                                                    Concentration at which colony                                                 formation was inhibited by                                                    greater than 50 percent                                                       Cell line                                                           Compound    BJAB       Raji      C85-5C                                       ______________________________________                                        (2'-5')A(pA).sub.2                                                                        150 -300 μM                                                                            300 μM                                                                              ND                                           (2'-5')A(pA).sub.3                                                                         150 μM   50 μM                                                                              ND                                           (2'-5')3'dA(p3'dA).sub.2                                                                  >300 μM >300 μM                                                                              ND                                           (3'-5')A(pA).sub.2                                                                        >300 μM >300 μM                                                                              ND                                           IFN-α ND         ND        >1000 U/ml                                   IFN-β  >500 U/ml  >500 U/ml >1000 U/ml                                   ______________________________________                                         *Determined by colony formation in microtiter plates following treatment      with compound for 7 days.                                                     ND = not determined.                                                     

We claim:
 1. The oligomer of the formula: ##STR2## wherein m is 0,1,2 or 3 and n is 0,1,2,3 or
 4. 2. 3'-Deoxyadenylyl-(2'-5')3'-deoxyadenylyl-(2'-5')3'-deoxyadenosine triphosphate being a compound of claim
 1. 3. 3'-Deoxyadenylyl-(2'-5')3'-deoxyadenylyl-(2'-5)-3'-deoxyadenosine diphosphate being a compound of claim
 1. 4. 3'-Deoxyadenylyl-(2'-5')3'deoxyadenylyl-(2'-5')-3'deoxyadenosine phosphate being a compound of claim
 1. 5. 3'-Deoxyadenylyl-(2'-5')3'-deoxyadenylyl-(2'-5')-3'-deoxyadenosine, being a compound of claim
 1. 6. An anti-viral composition which comprises an anti-Epstein Barr virus effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.
 7. An anti-viral composition effective against Epstein Barr virus which comprises an anti-viral effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.
 8. An anti-viral composition effective against Epstein Barr virus which comprises an anti-viral effective amount of a compound of claim 2,3,4 or 5 and a pharmaceutically acceptable carrier.
 9. A method of controlling viral infection which comprises administering to an Epstein Barr virus infected subject at anti-viral effective amount of a compound of claim
 1. 10. A method of inhibiting transformation of Epstein Barr virus which comprises administering to a subject infected with said virus an inhibitorily effective amount of a compound of claim
 1. 11. A method of inhibiting transformation of Epstein Barr virus which comprises administering to a subject infected with said virus an inhibitorily effective amount of a compound of claim 2,3,4 or
 5. 